Amino acid sequence of a novel heat-stable enterotoxin produced by a yst gene-negative strain of Yersinia enterocolitica

Summary A novel heat-stable enterotoxin (designated Y-STb) was isolated and purified to homogeneity from the culture supernatant of a pathogenic but yst gene-negative strain of Yersinia enterocolitica. The amino acid sequence of the toxin was determined to be Lys-Ala-Cys-Asp-Thr-Gln-Thr-Pro-Ser-Pro-Ser-Glu-Glu-Asn-Asp-Trp-Cys-Cys-Glu- Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys. Y-STb was 20-fold more potent (minimum effective dose in the suckling mouse assay was 0.35 pmol) than the previously documented heat-stable enterotoxin (Y-STa) which is produced by yst gene-positive strains of Y. enterocolitica and has a minimum effective dose of 7.8 pmol. The sequence of Y-STb is different from that of Y-STa in the N-terminal half (1–17), but quite similar in the C-terminal half (18–30). To elucidate the effect of 13 amino acid substitutions in Y-STb on enhancing the toxicity, several short analogs of Y-STb were synthesized and their toxicities were compared in the suckling mouse assay. The enhanced enterotoxicity could be ascribed to the addition of a tryptophan residue at the N-terminus of the ST toxic domain which is the minimum structure essential for toxic activity; the presence of an aspartic acid residue at the same position caused a decrease in toxicity.     

Enantioselective resolution of Rac‐terbutaline and evaluation of optically pure R‐terbutaline hydrochloride as an efficient anti‐asthmatic drug

Terbutaline is a β₂‐adrenoceptor agonist for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Among the two isomers of terbutaline (TBT 2), R‐isomer was found to be the potent enantiomer in generating therapeutic effect, while S‐isomer has been reported to show side effects. In this study, R‐terbutaline hydrochloride (R‐TBH 6) was synthesized through chiral resolution from the racemic terbutaline sulfate (rac‐TBS 1) with 99.9% enantiomeric excess (ee) in good overall yield (53.6%). Further, R‐TBH 6 nebulized solution was prepared in half dosage of Bricanyl®, which is a marketed product of racemic terbutaline and evaluated in vitro aerosol performance and in vivo anti‐asthmatic effect on guinea pigs via. pulmonary delivery. From the investigation, it is evident that R‐TBH 6 nebulized solution of half dosage performed similar fine aerosol characteristics and anti‐asthmatic effect with Bricanyl®.     

A novel post‐ligation thioesterification device enables peptide ligation in the N to C direction: synthetic study of human glycodelin

Human glycodelin consists of 162 amino acid residues and two N‐linked glycans at Asn²⁸ and Asn⁶³. In this study, we synthesized it by a fully convergent strategy using native chemical ligation (NCL) in N to C direction. The four peptide segments corresponding to 1–31, 32–65, 66–105 and 106–162 sequences were synthesized by 9‐fluorenylmethoxycarbonyl based solid‐phase peptide synthesis. At the C‐terminus of the second segment, N‐ethyl‐S‐acetamidomethyl‐cysteine was attached as a post‐ligation thioesterification device. The N‐terminal two segments were condensed by the homocysteine‐mediated NCL at Leu‐Met site, and the product was methylated to convert homocysteine to methionine. After deprotection of acetamidomethyl group on the N‐ethylcysteine residue, the peptide was thioesterified by N‐alkylcysteine‐assisted method. The product was then ligated with the C‐terminal half, which was obtained by the NCL of third and fourth segments, to give the full‐length glycodelin. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Amphibian Pleurodeles waltl Fibronectin: cDNA Cloning and Developmental Expression of Spliced Variants

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3′ region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.     

Non-coordinate regulation of RNA synthesis in escherichia coli exposed to 0°C

A non-coordinate mode of regulation of RNA synthesis is observed inEscherichia coli cells during exposure to 0°C. The stable RNA synthesis is preferentially inhibited with simultaneous accumulation of messenger RNA. The species of RNA synthesized at 0°C was determined by several criteria such as sedimentation value in sucrose gradients, DNA-RNA hybridization, half life measurements, protein synthesizing capacity and its functional rate of decay. The mode of regulation of RNA synthesis at 0°C is unique and is distinct from the non-coordinate regulation observed during amino acid starvation under stringent control.     

Intermolecular amination of allylic and benzylic alcohols leads to effective inhibitions of acetylcholinesterase enzyme and carbonic anhydrase I and II isoenzymes

In this study, we aimed to determine the inhibition effects of novel synthesized sulfamates ( 2a–g ), sulfonamides ( 3b–f ), carbonyl sulfonamides ( 3h and i ), and carbonyl sulfamates ( 4h and 4i ), which were tested against two human cytosolic carbonic anhydrase I and II isozymes (hCA I and II) and acetylcholinesterase (AChE) enzyme. For inhibition properties of allylic sulfamates, the half maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) were calculated for each novel compounds. The allylic sulfamates showed that K_i values are in the range of 187.33–510.31 pM for hCA I, 104.22–290.09 pM against hCA II, and 12.73–103.63 pM against AChE. The results demonstrated that all newly synthesized compounds had shown effective inhibition against hCA I and II isoenzymes and AChE enzyme.     

Lytic β-1,3 Glucanase from Arthrobacter: Pattern of Action

The action pattern of lytic β-1,3 glucanase (glucanase I) from Arthrobacter which liberates predominantly laminaripentaose from various β-glucans has been studied. The enzyme was not active on short linear laminaridextrins, but was active on an enzymatically synthesized, linear β-1,3 oligoglucan preparation. Any intactness of the glucose residues of the chain ends of a substrate did not seem to be necessary for the action of the enzyme. The results of determination of laminaripentaose during a relatively early phase of the reaction suggested that about half of the reducing power liberated in the medium might be explained by the formation of the sugar. It seems that the formation of laminaripentaose relates to the initial attack of glucanase 1 on β-1,3 glucan chains.     

Changes in Urease Activity in Apple Trees as Related to Urea Applications

Changes in urease (E.C.3.5.1.5.) were followed during the growth of 1-year-old MM 106 and 9-year-old Golden Delicious apple trees (Malus pumila Rehd.). Urease was found in leaves, roots, and bark with actively growing tissues containing more activity than senescing tissues. The urease activity in the leaves declined steadily during leaf senescence but abscised leaves still contained about half of their initial urease activity. In the bark the urease activity changed only slightly. Urease activities in the leaves and bark of apple trees were always greater in those trees which had received an application of urea. In senescing apple leaves, urea induced a rapid increase in urease activity. The changes in total activity and specific activity of urease were parallel and suggests that urease was synthesized de novo. After urease activity reached a maximum, a rapid decline occurred. Urease was inhibited by low concentrations of ammonia and this decline may be due to product inhibition.     

Co-polymerization of MTPC (methylene tri p-cresol) and m-cresol using CiP (Coprinus cinereus peroxidase) to improve the dissolution characteristics of the enzyme-catalyzed polymer

MTPC (Methylene tri p-cresol) and m-cresol were copolymerized by Coprinus cinereus peroxidase in aqueous acetone. Although MTPC did not dissolve completely in the aqueous acetone, copolymerization was achieved owing to the radical transfer between solute and solid surface. Various polymerized products with different molecular weights and hydroxyl values were synthesized depending upon reaction compositions (ratio of MTPC to m-cresol and buffer to acetone). Poly(MTPC–m-cresol), a copolymer of MTPC and m-cresol, was mixed with a diazonaphthoquinone derivative to form a new type of photoresist, a thin film of which was formed on a silicon wafer and immersed in alkaline solution (tetramethylammonium hydroxide) to measure speed of dissolution. Poly(MTPC–m-cresol), with higher hydroxyl value (over 80%), showed remarkably improved dissolution characteristics (dark loss in alkaline solution decreased by almost half), which is prerequisite for sensitive photoresist polymer.     

Anticonvulsant properties of spirohydantoins derived from optical isomers of camphor

Natural camphor exists as thed(+) form but thel(–) form has been synthesized. Replacement of the keto group on carbon 2 of each form with a hydantoin moiety led to only one spirohydantoin derivative. Bothd andl derivatives were synthesized. Both forms and their racemic mixture were tested in vivo for toxicity and behavioral effects in mice. A dose of 100 mg/kg of thed form was not toxic: mice showed normal grooming and exploratory behavior; thel form induced hunched posture, body jerks and myoclonic manifestations followed by quiescence. Thedl form showed intermediate effects. Challenge with the convulsant pentylenetetrazol (Metrazol) 2 hr after treatment with placebo or the camphor spirohydantoins produced seizure manifestations in all controls, in half of the subjects pretreated with thed-camphor derivative, in none of those pretreated with thel derivative and an intermediate response in those pretreated with racemic mixture. Thus, a spirohydantoin moiety added to camphor conferred strong anticonvulsive properties on thel form and modest ones on thed form; thed form did not seem to antagonize thel form.     

Protein Metabolism in the Domestic Fowl

The metabolic half lives of glycine in the tissue-proteins of the rooster were determined by single oral dose of 2-C¹⁴ glycine before measuring the amount of synthesized glycine in the rooster by ?constant pool? method. The specific activity of glycine originated from the purine ring of uric acid showed the highest value for 5 hrs. after administration, following rapid decrease until 7 days, thereafter slower one.

Although the specific activity of glycine in the tissue protein (serum and liver) decreased exponentially, its trend was not distinct in the pectoral muscle, and in the early period its decrease seemed to be considerably fast (t_1/2 about 6 days).

The specific activities of glycine in the serum protein were always higher than those in the liver protein. The metabolic half livers obtained were as follows. Liver: Faster 2 days, slower 11 days. Serum: faster 2 days, slower 11 days. Pectoral muscle: faster 6 days, slower 30 days. Recovery of C¹⁴ into 4-C and 5-C in the purine ring of excreted uric acid during 24 hours after the administration of isotope was about 24%.     

Individual messenger RNA half lives in Saccharomyces cerevisiae.

Summary We have measured the decay half-life of functional messenger RNA (mRNA) for some thirty different proteins in the yeast Saccharomyces cerevisiae. Production of newly synthesized mRNA was halted by raising the temperature of a culture of a temperature-sensitive mutant, ts 136. Aliquots of this culture were pulsed-labelled with [³⁵S]-methionine at various times after the temperature shift and the radioactive proteins separated on the two-dimensional gel electrophoresis system of O'Farrell. We find a range in the decay half lives of individual mRNA species which varies from 3.5 min to greater than 70 min. We find three general classes of decay curves, (a) simple exponential (first order); some of these showed a shoulder before onset of exponential decay; (b) bi-component or multi-component concave upward; (c) initial stimulation of rate of mRNA synthesis, followed by virtually undetectable decay.     

Copper(I)-catalysed synthesis of symmetrical perfluoroterphenyl analogues; fluorescence,antioxidant and molecular docking studies

Pentafluoroaryl analogues have been found to exhibit para specific nucleophilic aromatic substitution (S_NAr). Herein, we describe the use of S_NAr chemistry to create luminous perfluorinated symmetrical terphenyls. Both of S_NAr chemistry and copper(I)-catalysed decarboxylative cross-coupling were applied for the synthesis of the perfluorinated symmetrical terphenyls in high yields from the corresponding derivatives of aryl iodide and potassium salt of fluorobenzoate. A series of perfluorinated symmetrical terphenyls with different para alkoxy chains were synthesized. The synthesized perfluorinated terphenyl adducts were confirmed via elemental analysis, Fourier-transform infrared (FTIR), proton (¹H) carbon-13 (¹³C) and fluorine-19 (¹⁹F) nuclear magnetic resonance (NMR) spectra. The absorbance and fluorescence spectra showed solvatochromic activities. The new synthesized fluoroterphenyl hybrids were screened against antioxidant inspection over DPPH (2,2-diphenyl-1-picrylhydrazyl) performance, in assessment of vitamin C and butylated hydroxytoluene (BHT) as standard drugs exposed that fluoroterphenyl hybrid covering decyl hydrocarbons exhibited highest effectiveness through half maximal inhibitory concentration (IC₅₀) values of 21.74 μg/ml. Additionally, molecular docking procedures of the synthesized fluoroterphenyl hybrids were employed by using protein data bank (PDB ID: 5IKQ). The docking simulation displayed convenient and recognized findings with the antioxidant examination.     

Synthesis and Antibacterial Activity Evaluation of Imidazole Derivatives Containing 6-Methylpyridine Moiety

A series of 2-cyclopropyl-5-(5-(6-methylpyridin-2-yl)-2-substituted-1H-imidazol-4-yl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazoles ( 15a – t and 16a – f ) were synthesized and their antibacterial activities were evaluated. More than half of the compounds showed moderate or strong antibacterial activity. Among them, compounds 15t (MIC=1–2 μg/mL) and 16d (MIC=0.5 μg/mL) showed the strongest antibacterial activities. Notably, compound 16d did not exhibit cytotoxicity in HepG2 cells and did not show hemolysis like the positive control compound Gatifloxacin. The results suggest that compound 16d should be further investigated as a candidate antibacterial agent.     

Chloro and bromo-pyrazole curcumin Knoevenagel condensates augmented anticancer activity against human cervical cancer cells: design,synthesis, in silico docking and in vitro cytotoxicity analysis

Abstract

With an endeavor to develop novel curcumin analogs as potential anti-cancer agents, we designed and synthesized a series of Knoevenagel condensates by clubbing pyrazole carbaldehydes at the active methylene carbon atom of the curcumin backbone. Molecular docking studies were carried out to target the proposed derivatives on human kinase β (IKKβ), a potential anti-cancer target. The chloro derivative displayed five hydrogen bond interactions with a docking score of ?11.874?kcal/mol higher than curcumin (docking score =??7.434?kcal/mol). This was supported by the fact that the propellant shaped derivatives fitted aptly into the binding pocket. Molecular simulations studies were also conducted on the lead molecule and the results figured out that the stable complexes were developed as the minimal deviations per residue of protein within the range of 0.11–0.92 Å. The screened compounds were synthesized, characterized and evaluated in vitro for cytotoxicity against cervical cancer cell line, HeLa using standard cell proliferation assay. Chloro derivative and bromo analog demonstrated IC₅₀ (half maximal inhibitory concentration) value of 14.2 and 18.6 µg/ml, respectively, significantly lower than 42.4 µg/ml of curcumin and higher than 0.008 µg/ml of paclitaxel. Induction of apoptosis was evaluated in the terms of cleavage of caspase-3 enzyme and they also exhibited 69.6 and 65.4% of apoptosis significantly higher than 19.9% induced by curcumin. In conclusion, chloro and bromo derivatives must be evaluated under a set of stringent in vitro and in vivo parameters for translating in to a clinically viable product.

Communicated by Ramaswamy H. Sarma     

Green synthesis of tellurium-doped SnO2 nanoparticles with sulfurized g-C3N4: Insights into methylene blue photodegradation and antibacterial capability

The construction of SnO₂ nanoparticles (NPs), specifically Te-doped SnO₂ NPs, using a simple and economical co-precipitation technique has been thoroughly described in this work. NH₃ served as the reducing agent in this procedure, whilst polyethylene glycol served as the capping agent. The primary goals of our work were to investigate the physicochemical properties of the synthesized SnO₂ NPs and assess their potential use as antibacterial agents and photocatalysts. Scanning electron microscopy–energy dispersive X-ray, ultraviolet light, Fourier transform infrared spectroscopy, X-ray diffraction (XRD), and other analytical techniques were used to thoroughly analyze the NPs. Based on the full width at half maximum of the most noticeable peaks in the XRD spectrum, the Debye–Scherrer equation was used to calculate the crystallite sizes, which indicated the presence of a single tetragonal SnO₂ phase. Particularly noteworthy was the exceptional photocatalytic activity of graphene-assisted Te-doped SnO₂ NPs, achieving an impressive decomposition efficiency of up to 98% in the photo-oxidation of methylene blue. Furthermore, our investigation delved into the antibacterial attributes of the synthesized SnO₂ NPs against Escherichia coli and Staphylococcus aureus, demonstrating inhibitory effects on both bacteria strains. This suggests potential applications for these NPs in various environmental and medical contexts.     

Synthesis of Signaling N-Acyl-Homoserine-Lactones Participating in Quorum Sensing Regulation in Rhizospheric and Soil-Borne Bacteria Pseudomonas and Xanthomonas

Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonasand Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize these compounds. In at least a half of the isolated P. aureofaciens andP. aeruginosa strains, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri,P. geniculata,and P. putidastrains. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.     

Stability of some microginins synthesized by the cyanobacterium Woronichinia naegeliana (Unger) Elenkin

Microginins are linear oligopeptides synthesized by cyanobacteria. The literature data on their characteristics are scant. This study examined the influence of abiotic factors including pH, temperature, visible and ultraviolet radiation on the stability of the microginins FR3 (MG‐FR3), FR4 (MG‐FR4) and 757 (MG‐757) synthesized by Woronichinia naegeliana. In alkaline conditions (pH 9) only the concentration of MG‐757 was reduced significantly, by 14.3%. The tested microginins were stable at room temperature (half‐life 7–17 weeks). Boiling for one hour caused 26.1% decomposition of MG‐FR4 and 26.8% decomposition of MG‐757; MG‐FR3 was not significantly affected. Under visible radiation the initial content of MG‐FR4 declined 23.0%, but MG‐FR3 and MG‐757 proved insensitive to it. Treatment with a high dose of UV radiation (36 μmol m^?2 s^?1) caused the tested microginins to degrade by 13.8% to 21.4%. The study showed these microginins to be oligopeptides of high stability, the most stable of them being MG‐FR3.     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.